CN110760617A - Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild virus - Google Patents
Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild virus Download PDFInfo
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Abstract
The invention relates to the technical field of animal virus detection in the field of veterinarians, and particularly discloses a real-time fluorescent PCR primer probe combination and a kit for detecting African swine fever virus wild viruses; the PCR method based on the kit can specifically detect the African swine fever virus wild virus. When the fluorescent PCR kit is matched with the existing fluorescent PCR of p72 for use, the fluorescent PCR kit can also be used as a method for distinguishing African swine fever virus wild viruses and MGF360-505R deletion attenuated vaccine strains, thereby providing an important tool for detecting the African swine fever virus wild viruses and infected animals thereof, and being beneficial to control and purification of the African swine fever virus.
Description
Technical Field
The invention relates to the technical field of animal virus detection in the field of veterinarians, in particular to a real-time fluorescent PCR primer probe combination and a kit for detecting African swine fever virus.
Background
African Swine Fever (ASF) is a virulent, hemorrhagic infectious disease of pigs caused by African Swine Fever Virus (ASFV); virulent strains can kill domestic pigs within about 5-14 days of infection, with mortality rates approaching 100% and difficulty in being killed. The disease is an animal epidemic disease prescribed by the world animal health Organization (OIE) and is legally reported. After the first African swine fever epidemic situation occurs in China in 8 months in 2018, the method has made a very heavy hit on the pig industry in China.
Since the discovery of the African swine fever virus for more than 100 years, only the countless countries of Spain, Brazil, etc. rely on the suicide strategy to successfully control the African swine fever. However, the raising quantity of live pigs in China is huge, the live pigs are only fed by means of killing, the cost is too high, and safe and effective vaccines are urgent requirements for controlling epidemic situation of African swine fever in China. However, the conventional vaccine development strategies including inactivated vaccines, natural attenuated or passage attenuated vaccines, and the like), novel adjuvants, subunit vaccines, and the like are not successful.
Foreign studies indicate that the deletion of fragments of the African swine fever virus genomes 178643-182578 causes the deletion of reading frames of MGF505-11L, MGF100-1L, 17L18L ASFV G ACD01870, I9R and I10L I11L, can lead to the significant reduction of virulence of virus, and can generate effective protection as a low-virulent vaccine. The research unit in China also proves that the African swine fever virus gene deletion strain obtained by carrying out MGF360-505R gene deletion on the African swine fever virus with the genotype I or carrying out combined deletion of CD2V and MGF360-505R genes not only is fully weakened, but also is very safe as a vaccine.
At present, the African swine fever virus MGF360-505R deletion attenuated vaccine strain has shown good safety and protection effect; however, once the vaccine is clinically applied, the existing detection technology based on the African swine fever virus P72 gene and the like cannot distinguish the wild type strain virus from the vaccine virus, so that the detection of the vaccine virus and the wild type virus is positive. This would greatly interfere with the detection of the African swine fever virus wild virus after the use of the attenuated vaccine, thereby adversely affecting the control of the disease.
Therefore, the development of a detection kit and a method for specifically detecting African swine fever virus after MGF360-505R deletion attenuated vaccine is used is urgently needed. Therefore, after the attenuated vaccine is used, wild virus infected animals are specifically detected and screened, thereby providing an important tool for preventing and controlling the disease.
Disclosure of Invention
The invention aims at the requirements and technical problems that after MGF360-505R deletion attenuated vaccine is applied to preventing and controlling African swine fever, the elimination of African swine fever virus wild virus infected animals needs to be detected; the real-time fluorescent PCR primer probe combination and the kit for detecting the African swine fever virus wild virus are provided, so that an important tool is provided for detecting the African swine fever virus wild virus and infected animals thereof under the using background of vaccines, and the control and purification of the African swine fever virus wild virus are facilitated.
In order to realize the purpose, the invention designs a real-time fluorescent PCR primer probe combination for detecting African swine fever virus wild virus, which comprises a fluorescent PCR specific primer and a fluorescent PCR probe; the nucleotide sequence of the fluorescent PCR specific primer is as follows:
the fluorescent PCR primers are:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3',
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3',
y is C/T; m is A/C, R is A/G;
the nucleotide sequence of the fluorescent PCR probe is as follows: 5'-TAAACTACTCCGTGAAAC-3', and the fluorescent PCR probe has a nucleotide sequence with a 5 'end combined with a fluorescent reporter group and a 3' end combined with a fluorescent quencher group.
Further, the fluorescence reporter group is FAM, and the fluorescence quencher group is BHQ.
The invention also provides a kit for detecting the African swine fever virus wild strain, which comprises the fluorescent PCR primer probe combination. Preferably, the method also comprises qPCRProbe MasterMix.
The method for detecting the African swine fever virus wild strain by using the kit comprises the following steps:
1) establishing a standard curve
a. Preparation of a standard substance:
the African swine fever virus genome DNA is taken as a template, and the following PCR primers are used:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3',
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3',
y is C/T; m is A/C, R is A/G;
and carrying out PCR amplification to obtain a PCR product, wherein the sequence of the PCR product is shown as SEQ ID NO: 4, connecting the PCR product with a pMD18-T vector to construct a positive plasmid pMGF-505-2R, namely a standard substance;
b. establishment of a Standard Curve
Making the positive plasmid into a standard template, and detecting by using fluorescence PCR (the nucleotide sequence of the primer is that an upstream primer is 5 '-TCTGCGTCAACTACCYCG-3', a downstream primer is 5 '-ATMGTCYTYACTTTCRTC-3', Y is C/T, M is A/C, R is A/G, the nucleotide sequence of a fluorescence PCR probe is 5'-TAAACTACTCCGTGAAAC-3', the 5 'end of the nucleotide sequence of the fluorescence PCR probe is combined with a fluorescence reporter group, and the 3' end of the nucleotide sequence of the fluorescence PCR probe is combined with a fluorescence quenching group); and establishing a standard curve by utilizing the functional relation between the Lg value of the genome copy number and the Ct value of the detection:
lg (gene copies/. mu.L) — 0.2449x +12.348, R2 ═ 0.9972, wherein Y is an Lg value; x is Ct value;
2) testing of samples to be tested
a. Fluorescent PCR
Collecting a sample to be detected and extracting a genome of the sample; the following fluorescent PCR primers and probes were used:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3', downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3', Y is C/T; m is A/C, R is A/G;
the nucleotide sequence of the fluorescent PCR probe is as follows: 5'-TAAACTACTCCGTGAAAC-3', and the fluorescent PCR probe has a nucleotide sequence with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end
Performing fluorescence PCR amplification, and collecting a fluorescence signal to obtain Ct;
b. determination of detection result
If Ct is less than 40, the result is positive, the sample to be detected contains African swine fever virus wild strain (the lower the Ct value is, the higher the virus content of the wild virus is); bringing the Ct value of the sample to be detected into a standard working curve, namely judging the virus content of the African swine fever virus wild strain in the sample to be detected;
or if Ct > is 40 or no amplification curve, the wild virus is not contained in the sample to be detected or the wild virus content is extremely low and cannot be detected (further inspection shows that the virus contained in the sample is MGF360-505R deletion attenuated vaccine strain because the existing fluorescent PCR of p72 is positive).
The invention has the beneficial effects that:
the real-time fluorescent PCR primer probe combination for detecting the African swine fever virus can detect the African swine fever virus wild virus containing a target sequence, but the African swine fever virus MGF360-505R deletion attenuated vaccine strain can not be detected due to the deletion of the target sequence. Therefore, the PCR method based on the kit can specifically detect the African swine fever virus wild virus. When the fluorescent PCR kit is matched with the existing fluorescent PCR of p72 for use, the fluorescent PCR kit can also be used as a method for distinguishing African swine fever virus wild viruses and MGF360-505R deletion attenuated vaccine strains, thereby providing an important tool for detecting the African swine fever virus wild viruses and infected animals thereof, and being beneficial to control and purification of the African swine fever virus.
Drawings
FIG. 1 is a linear relationship chart between the log value of plasmid gene copy number and Ct value of fluorescence PCR detection, which is plotted after fluorescence PCR detection is performed on each dilution sample of the positive plasmid pMGF _505-2R of example 2; wherein the plasmid copy numbers are respectively 2 × 1010、2×109、2×108、2×107、2×106、2×105、2×104、2×103、2×102、2×101、2×100Copy/. mu.L;
FIG. 2 is the amplification curve of 30 parallel samples in the same experiment with copy number of the positive plasmid pMGF _505-2R of example 2;
FIG. 3 is a block flow diagram of the fluorescence PCR detection method for detecting African swine fever virus and distinguishing wild virus from MGF360-505R deleted attenuated vaccine strains of example 3.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Example 1
Screening of real-time fluorescent PCR primer probe combination for detecting African swine fever virus wild virus
According to a genome target sequence deleted by the attenuated vaccine, namely a genome 27942-35500bp sequence of the African swine fever virus Chinese epidemic strain Pig/CN/HLJ/2018, considering factors such as primer mismatching, Tm value and the like, designed primers and probes are respectively analyzed in an NCBI (national center for Biotechnology information) database through Blast software, and a primer pair and a probe sequence are required to cover all known African swine fever virus sequences in the database and have no obvious pairing with other sequences. Through analysis, the following results are found:
the sequences 5'-TCTGCGTCAACTACCTCG-3', 5'-TAAACTACTCCGTGAAAC-3' and 5'-GACGAAAGTGAGGACGAT-3' can match with the sequences of most African swine fever viruses, and can be used as a combination for detecting the African swine fever virus wild viruses, namely a primer pair (an upstream primer: 5'-TCTGCGTCAACTACCTCG-3' and a downstream primer: 5'-GACGAAAGTGAGGACGAT-3', and a probe 5'-TAAACTACTCCGTGAAAC-3').
When the sequence matched with the primer and the African swine fever virus is further analyzed, 16 bases at the position of the upstream primer are found, the sequence of partial virus is C, and therefore, the upstream primer is revised to be 5 '-TCTGCGTCAACTACCYCG-3'; 3, 7, 9 and 16 bases of the downstream primer, the sequence of partial virus is A, T, T, A respectively, and the downstream primer is modified to be 5 '-ATMGTCYTYACTTTCRTC-3' in order to cover all African swine fever viruses; the nucleotide sequence 5'-TAAACTACTCCGTGAAAC-3' of the probe can be used to cover all the African swine fever virus sequences known in databases. Therefore, a primer probe combination of the fluorescence PCR detection method of the African swine fever virus wild virus is established: namely, the fluorescent PCR primers are:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3' as shown in SEQ ID NO. 1;
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3' as shown in SEQ ID NO. 2;
y is C/T; m is A/C, R is A/G;
the nucleotide sequence of the fluorescent PCR probe is as follows: 5'-TAAACTACTCCGTGAAAC-3', as shown in SEQ ID NO.3, the 5 'end of the nucleotide sequence of the fluorescent PCR probe is combined with fluorescent reporter groups such as FAM, and the 3' end is combined with BHQ fluorescent quenching groups.
Then, taking the genome of the African swine fever virus as a template, and detecting the expression quantity of the genes by adopting a qPCR (quantitative polymerase chain reaction) kit; the reaction system comprises Mix 5 mu L, ROX 0.2.2 mu L, 10 mu M primers 0.25 mu L, 10 mu M probe 0.1 mu L, DNA template 2 mu L, and water supplement to 10 mu L; the negative control group replaces a sample to be detected with deionized water, and simultaneously sets genomes of the porcine pseudorabies virus and the classical swine fever virus as nonspecific controls;
fluorescent PCR reaction conditions: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 10s, annealing at 50 ℃ for 10s, extension at 60 ℃ for 30s, and collecting fluorescence signals at 60 ℃ for 45 cycles;
the fluorescent PCR detection result shows that the target sequence can be detected after 100 times of tests, and the Ct value is less than 25, which is equivalent to the current fluorescent detection method based on the P72 sequence. And the genomes of other viruses (such as porcine pseudorabies virus and classical swine fever virus) are used as templates and have no detection signal, and negative control has no detection signal, so that the fluorescent PCR can be used as a method for detecting the African swine fever virus wild virus: wherein:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3',
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3',
y is C/T; m is A/C, R is A/G;
the nucleotide sequence of the fluorescent PCR probe is as follows: 5'-TAAACTACTCCGTGAAAC-3', the 5 'end of the nucleotide sequence of the fluorescent PCR probe is combined with fluorescent reporter group such as FAM, and the 3' end is combined with BHQ fluorescent quenching group.
Example 2
The kit for detecting the African swine fever virus wild strain comprises the fluorescent PCR primer probe combination; in the present invention, primers and probes can be added directly to the commercial qPCR Probe MasterMix reagents. The method for detecting the African swine fever virus wild virus based on the kit construction comprises the following steps:
1) preparation of a standard substance:
taking African swine fever virus genome DNA as a template, and using fluorescent PCR primers:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3',
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3', Y is C/T; m is A/C, R is A/G,
performing PCR amplification to obtain a gene partial fragment (shown as SEQ ID NO: 4) with a product of 258bp, connecting the gene partial fragment with a pMD18-T vector, constructing a positive plasmid pMGF-505-2R containing a target fragment, and performing sequencing without mutation;
2) establishment of a standard curve:
2 x 10 to10Copy/. mu.L positive plasmid was diluted 10-fold in gradient to 1X 10 concentration10-1×102Copy/. mu.L, as a standard template, and by performing fluorescence PCR with reference to a reaction system and conditions in the "target gene screening", the functional relationship between the Lg value of the genome copy number and the Ct value detected is Lg (gene copies/. mu.L) — 0.2449x +12.348, and R2 ═ 0.9972, which indicates that the correlation is good;
3) sensitivity and repeatability experiment of kit for detecting African swine fever virus wild strain
Will be 1 × 1010Copy/. mu.L positive plasmid was diluted 10-fold in gradient to 1X 10 concentration10-1×102Copy/. mu.L, used as standard template for the qPCR reaction sensitivity test, each sample set for 3 replicates. The fluorescence PCR detection is carried out on each dilution sample of the positive plasmid pMGF-505-2R.
The results show that: the method is inaccurate when Ct > is 40, so the genome copy number corresponding to Ct <40 is taken as the detectable range of the method, and therefore, the method can detect 356 virus nucleic acid molecule copy numbers at the lowest, as shown in figure 1, and has higher sensitivity.
The result of the repeated experiments shows that about 8X 10 is taken7The copy/. mu.L positive plasmid is repeated for 30 times in 1 experiment, the experiment is repeated for 3 times, the repeatability of the method is evaluated through the Ct value variation coefficient (standard deviation/mean of repetition value) between groups, the result shows that 30 parallel sample amplification curves in the same experiment are basically coincident near a threshold line (figure 2), and the variation coefficients between groups and in groups are less than 5%, which indicates that the method has higher stability and repetitionAnd the method can be used for detecting the African swine fever virus genome DNA.
Example 3
The flow of the detection method of the kit for detecting the African swine fever virus wild strain and the judgment of the result (figure 3) comprise the following steps:
(1) extracting genome from sample to be detected
(2) Detection by fluorescence PCR:
the fluorescent PCR reaction system comprises Mix 5 mu L, ROX 0.2.2 mu L, 10 mu M fluorescent PCR primers 0.25 mu L respectively, 10 mu M fluorescent PCR probe 0.1 mu L, DNA template 2 mu L, and water supplement to 10 mu L;
fluorescent PCR reaction conditions: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 10s, annealing at 50 ℃ for 10s, extension at 60 ℃ for 30s, 45 cycles, and collecting fluorescence signals at 60 ℃;
(3) and (3) judging a detection result:
if the Ct is positive when the Ct is less than 40, the sample contains wild virus. And the lower the Ct value is, the higher the virus content of the wild virus is; if Ct > is 40 or no amplification curve, the sample does not contain wild virus or the wild virus content is extremely low to be undetectable; and further detection: the fluorescence PCR based on p72 is positive, which indicates that the virus contained in the sample is MGF360-505R deletion attenuated vaccine strain.
Example 4
The kit for detecting the African swine fever virus wild strain is applied to the detection of clinical samples:
the detection is carried out by applying fluorescent PCR to the genome (positive in African swine fever virus P72 fluorescent PCR detection) of 10 clinical positive samples (4 serum samples and 6 tissue samples) infected by African swine fever virus, 3 clinical healthy pig tissue samples (4 serum samples and 6 tissue samples) infected by porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus.
The reaction system of the fluorescence PCR is that Mix 5 mu L, ROX 0.2.2 mu L, 10 mu M fluorescence PCR primer is 0.25 mu L, 10 mu M fluorescence PCR probe is 0.1 mu L, DNA template is 2 mu L, and water is supplemented to 10 mu L;
fluorescent PCR reaction conditions: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 10s, annealing at 50 ℃ for 10s, extension at 60 ℃ for 30s, 45 cycles, and collecting fluorescence signals at 60 ℃;
the detection result of the fluorescence PCR shows that the Ct of all African swine fever infection positive samples is less than 25; the Ct detected by all the healthy pig tissue samples is more than 42 or has no obvious amplification curve; ct of porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus infected porcine tissue samples detected by the fluorescent PCR is more than 40. The method can be used for detecting the African swine fever virus wild viruses.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
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Claims (5)
1. A real-time fluorescent PCR primer probe combination for detecting African swine fever virus wild virus is characterized in that: it comprises a fluorescent PCR specific primer and a fluorescent PCR probe; the nucleotide sequence of the fluorescent PCR specific primer is as follows:
the fluorescent PCR primers are:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3',
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3',
y is C/T; m is A/C, R is A/G;
the nucleotide sequence of the fluorescent PCR probe is as follows: 5'-TAAACTACTCCGTGAAAC-3', and the fluorescent PCR probe has a nucleotide sequence with a 5 'end combined with a fluorescent reporter group and a 3' end combined with a fluorescent quencher group.
2. The real-time fluorescent PCR primer probe combination for detecting African swine fever virus wild virus according to claim 1, which is characterized in that: the fluorescence reporter group is FAM, and the fluorescence quenching group is BHQ.
3. A kit for detecting wild type strains of African swine fever virus is characterized in that: comprising the fluorescent PCR primer probe combination of claim 1.
4. The kit of claim 3, wherein: also included is qPCR Probe MasterMix.
5. The method for detecting the African swine fever virus wild-type strain by using the kit of claim 3, which is characterized in that: the method comprises the following steps:
1) establishing a standard curve
a. Preparation of a standard substance:
the African swine fever virus genome DNA is taken as a template, and the following PCR primers are used:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3',
a downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3',
y is C/T; m is A/C, R is A/G;
and carrying out PCR amplification to obtain a PCR product, wherein the sequence of the PCR product is shown as SEQ ID NO: 4, connecting the PCR product with a pMD18-T vector to construct a positive plasmid pMGF-505-2R, namely a standard substance;
b. establishment of a Standard Curve
Making the positive plasmid into a standard template, and detecting by using fluorescence PCR (the nucleotide sequence of the primer is that an upstream primer is 5 '-TCTGCGTCAACTACCYCG-3', a downstream primer is 5 '-ATMGTCYTYACTTTCRTC-3', Y is C/T, M is A/C, R is A/G, the nucleotide sequence of a fluorescence PCR probe is 5'-TAAACTACTCCGTGAAAC-3', the 5 'end of the nucleotide sequence of the fluorescence PCR probe is combined with a fluorescence reporter group, and the 3' end of the nucleotide sequence of the fluorescence PCR probe is combined with a fluorescence quenching group); and establishing a standard curve by utilizing the functional relation between the Lg value of the genome copy number and the Ct value of the detection:
-0.2449x +12.348, R2 0.9972, wherein Y is Lg value; x is Ct value;
2) testing of samples to be tested
a. Fluorescent PCR
Collecting a sample to be detected and extracting a genome of the sample; the following fluorescent PCR primers and probes were used:
an upstream primer: 5 '-TCTGCGTCAACTACCYCG-3', downstream primer: 5 '-ATMGTCYTYACTTTCRTC-3', Y is C/T; m is A/C, R is A/G;
the nucleotide sequence of the fluorescent PCR probe is as follows: 5'-TAAACTACTCCGTGAAAC-3', and the fluorescent PCR probe has a nucleotide sequence with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end
Performing fluorescence PCR amplification, and collecting a fluorescence signal to obtain Ct;
b. determination of detection result
If Ct is less than 40, the result is positive, which indicates that the sample to be detected contains African swine fever virus wild strain; bringing the Ct value of the sample to be detected into a standard working curve, namely judging the virus content of the African swine fever virus wild strain in the sample to be detected;
or if Ct > is 40 or no amplification curve, indicating that the sample to be detected does not contain wild virus or the wild virus content is extremely low to be undetectable.
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| CN110872637A (en) * | 2019-12-04 | 2020-03-10 | 深圳海关动植物检验检疫技术中心 | Reagent for identifying African swine fever gene deletion vaccine, detection method and application |
| CN111304371A (en) * | 2020-04-23 | 2020-06-19 | 广东省农业科学院动物卫生研究所 | Locked nucleic acid probe fluorescent quantitative PCR detection composition, detection method and detection kit for African swine fever virus wild strain |
| CN111500790A (en) * | 2020-06-10 | 2020-08-07 | 四川农业大学 | Primer probe set for detecting African swine fever by fluorescent quantitative PCR and application thereof |
| CN111575404A (en) * | 2020-05-08 | 2020-08-25 | 中国兽医药品监察所 | Gene chip for differential diagnosis of swine fever wild virus and vaccine thereof, African swine fever virus and detection method |
| CN111676327A (en) * | 2020-07-21 | 2020-09-18 | 河南省农业科学院 | Dual fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for African swine fever virus wild virus infection and gene deletion strain |
| CN111690775A (en) * | 2020-06-24 | 2020-09-22 | 哈尔滨元亨生物药业有限公司 | African swine fever virus fluorescence PCR rapid detection kit |
| CN113025629A (en) * | 2020-05-01 | 2021-06-25 | 中国农业科学院兰州兽医研究所 | Attenuated African swine fever virus strain with gene deletion and application thereof |
| CN113502352A (en) * | 2021-07-01 | 2021-10-15 | 华中农业大学 | EMA-ddPCR primer and probe for detecting infectious ASFV and application |
| CN114107228A (en) * | 2021-11-11 | 2022-03-01 | 中国农业科学院兰州兽医研究所 | Construction of attenuated African swine fever virus strain with twelve genes deleted and application of attenuated African swine fever virus strain as vaccine |
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| CN110872637A (en) * | 2019-12-04 | 2020-03-10 | 深圳海关动植物检验检疫技术中心 | Reagent for identifying African swine fever gene deletion vaccine, detection method and application |
| CN111304371A (en) * | 2020-04-23 | 2020-06-19 | 广东省农业科学院动物卫生研究所 | Locked nucleic acid probe fluorescent quantitative PCR detection composition, detection method and detection kit for African swine fever virus wild strain |
| CN113025629A (en) * | 2020-05-01 | 2021-06-25 | 中国农业科学院兰州兽医研究所 | Attenuated African swine fever virus strain with gene deletion and application thereof |
| CN111575404A (en) * | 2020-05-08 | 2020-08-25 | 中国兽医药品监察所 | Gene chip for differential diagnosis of swine fever wild virus and vaccine thereof, African swine fever virus and detection method |
| CN111575404B (en) * | 2020-05-08 | 2023-03-14 | 中国兽医药品监察所 | Gene chip for differential diagnosis of swine fever wild virus and vaccine thereof, african swine fever virus and detection method |
| CN111500790A (en) * | 2020-06-10 | 2020-08-07 | 四川农业大学 | Primer probe set for detecting African swine fever by fluorescent quantitative PCR and application thereof |
| CN111690775A (en) * | 2020-06-24 | 2020-09-22 | 哈尔滨元亨生物药业有限公司 | African swine fever virus fluorescence PCR rapid detection kit |
| CN111676327A (en) * | 2020-07-21 | 2020-09-18 | 河南省农业科学院 | Dual fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for African swine fever virus wild virus infection and gene deletion strain |
| CN113502352A (en) * | 2021-07-01 | 2021-10-15 | 华中农业大学 | EMA-ddPCR primer and probe for detecting infectious ASFV and application |
| CN113502352B (en) * | 2021-07-01 | 2022-02-01 | 华中农业大学 | EMA-ddPCR primer and probe for detecting infectious ASFV and application |
| CN114107228A (en) * | 2021-11-11 | 2022-03-01 | 中国农业科学院兰州兽医研究所 | Construction of attenuated African swine fever virus strain with twelve genes deleted and application of attenuated African swine fever virus strain as vaccine |
| CN114107228B (en) * | 2021-11-11 | 2023-06-13 | 中国农业科学院兰州兽医研究所 | Construction of attenuated African swine fever virus strain with twelve genes deleted and application of attenuated African swine fever virus strain as vaccine |
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